Development of methods for analyzing a high molecular polymer such as a DNA and a protein using a nanometer-sized pore called as a nanopore has been advanced. It has been technically difficult to open the nanopore, however it has been realized to open the nanopore for the first time in a biological field by introducing an ion channel to a lipid bilayer membrane (Non Patent Literature 1). Moreover, a measurement method according to a patch-clamp method used in an ion channel measurement in a living body has been also developed as a measurement method using the nanopore. Next, opening the nanopore by utilizing a semiconductor process has been tried, and a method by an ion beam (Non Patent Literature 2) and a method by an electron beam (Non Patent Literature 3) have been developed.
As the nanopore can be created, the methods for analyzing a polymer molecule such as a DNA and a protein have been developed. Principal methods necessary for analysis with nanopore are the following two methods.
1. Detection method: those for detecting a physical change of a polymer molecule while the polymer molecule is transferred through the nanopore
2. Polymer driving method: those for transferring a polymer molecule to make the polymer molecule pass through the nanopore
As the detection method, a current blockade method, a tunneling current method, and a capacitance measurement method are known. The current blockade method is a method of detecting blocking effect caused by partially blocking a nanopore opening with a polymer molecule (Non Patent Literature 4). A specific structure is a structure obtained by separating a space into two by a membrane having a nanopore, filling each space with a liquid containing an ion, and disposing an electrode in each space. When a given voltage is applied to the electrodes, the ion is driven through the nanopore and thus a current flows (ionic current). When a charged polymer molecule is present, the polymer molecule is also attracted to one side and transferred through the nanopore by an electric potential difference. Since the nanopore opening is partially blocked at the time of the transfer, the ion becomes hard to flow through the nanopore and the amount of ionic current is lowered. The current blockade method is a method of analyzing the presence of a polymer molecule or a component of a polymer molecule by detecting the lowering of the electric current value. The extent of the resistance to flow of an ion is affected by the charged state of the polymer molecule and the interaction with a nanopore wall in addition to the area of the nanopore opening.
On the other hand, the tunneling current method is a method of analyzing the presence of a polymer molecule or a component of a polymer molecule by detecting a tunneling current flowing through a slight gap between an electrode for a tunneling current provided near the nanopore and the polymer molecule when the polymer molecule is transferred through the nanopore (Non Patent Literature 5 and Non Patent Literature 6).
The capacitance measurement method is a method of analyzing the presence of a polymer molecule or a component of a polymer molecule by detecting the change in the capacitance of a nanopore-having membrane caused by the partial blockage of nanopore during the transfer of the polymer molecule through the nanopore (Non Patent Literature 7).
Moreover, the polymer driving method includes a differential voltage driven method, an enzymatic driven method, and a force driven method. The differential voltage driven method is a method of transferring a charged polymer molecule according to an electric field gradient by disposing electrodes to two spaces separated by a membrane having a nanopore and applying voltages to the electrodes as described for the current blockade method. Examples of its advantages include that the differential voltage driven method can be realized by a simple structure and unnecessary load is not posed to the polymer molecule.
The enzymatic driven method is a method of transferring a polymer molecule by disposing an enzyme near the nanopore and utilizing the reaction of the polymer molecule with the enzyme. For example, there is a method of transferring a DNA nucleotide by nucleotide by disposing a DNA polymerase near the nanopore and causing to synthesize a double strand DNA in the case where the polymer molecule is a single strand DNA. It is necessary that the DNA be located near the DNA polymerase in the enzymatic driven method, and therefore it is preferred to collect the DNA near the nanopore using the differential voltage driven method in combination in view of improving efficiency.
The force driven method is a method of realizing the transfer of a polymer molecule by fixing the polymer molecule to a bead and transferring the bead by optical tweezers. It is necessary that an end of the polymer molecule where the bead is not attached be inserted in the nanopore in the force driven method, and the differential voltage driven method is adopted for the insertion.
Accordingly, the differential voltage driven method is used, as one providing a driving force to carry the sample to the nanopore opening, in combination with any of the methods.
The sample is carried to the nanopore opening via the following three transferring states and is transferred through the nanopore. The first is the transfer by diffusion, the second is the transfer by electrophoresis, and the third is the probability of accession of an end of a polymer molecule to the nanopore opening when the sample is a long chain (Non Patent Literature 8). The electric potential difference largely affects in the second and the third transfer states.
In the conventional analysis with nanopore, since the frequency of accession of a sample to the nanopore opening is lowered when the sample has a low concentration, the transfer frequency through the nanopore is also lowered, thereby causing a a problem that the throughput in the measurement is lowered.